This work was supported by R01 DC013339, R03 DC011373, and Ruth L. Kirschstein National Research Service Award T32 DC000014. A portion of the work was performed using the Monell&#160;Chemosensory Receptor Signaling Core, which is supported, in part, by funding from the NIH-NIDCD Core Grant P30 DC011735. The authors thank C. Sezille for help with data collection.

1. Culture of Hana3A Cells
<ol>
	<li>Prepare M10 media by supplementing minimum essential medium (MEM) with 10% (v/v) FBS.</li>
	<li>Culture Maintenance
	<ol>
		<li>Maintain cells in M10 media. NOTE: The expression vectors for RTP1L, RTP2, REEP1, and G&#945;olf confer puromycin resistance to Hana3A cells, but maintaining the cells with this antibiotic does not significantly affect assay results.</li>
		<li>Subculture at a ratio of 1:8 in 10 cm dishes every 2-3 days.</li>
		<li>Incubate at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Plating Cells for Transfection
<ol>
	<li>Aspirate media from a 100% confluent 10 cm dish of Hana3A cells.</li>
	<li>Wash cells by adding 10 ml PBS, swirling the dish, and aspirating the PBS.</li>
	<li>Add 3 ml of 0.05% trypsin/EDTA and wait for cells to dissociate (about 1 min).</li>
	<li>Inactivate trypsin by adding 5 ml M10 and break up cell clumps by triturating roughly 10x&#160;with a 10 ml pipette. Pipette carefully to avoid introducing air bubbles into the media.</li>
	<li>For each 96-well plate, transfer 1 ml of cells into a 15 ml conical tube, centrifuge at 200 x g for 5 min, and aspirate the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend the cells in 6 ml M10 per 1 ml of cells transferred in Step 2.5.</li>
	<li>Pipette 50 &#181;l of cells to each well of a 96-well plate and incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
3. Transfection of Olfactory Receptors
<ol>
	<li>Preparation of Plasmid DNA
	<ol>
		<li>Prepare plasmid DNA via an endotoxin-free protocol. NOTE: Use plasmid DNA preparation kits designated &#34;endotoxin-free,&#34; or add a phenol-chloroform extraction step to the plasmid DNA preparation protocol.</li>
		<li>Dilute DNA to a concentration of 100 ng/&#181;l in TE buffer.</li>
	</ol>
	</li>
	<li>Observe plated cells (Step 2.7) to ensure a proper confluency of approximately 30-50% per well and return to incubator. NOTE: While this confluency is not optimal for the lipid transfection reagent, a confluency of 30-50% at this step is optimal for measuring luciferase activity 24 hr after transfection.</li>
	<li>Preparation of Transfection Mix
	<ol>
		<li>Pipette RTP1S-pCI, M3-R-pCI, pCRE-luc, and pSV40-RL plasmids into MEM medium per the volumes detailed in Table 1 to make the Plasmid mix (volumes indicated are per 96-well plate).
		<table border="0" cellpadding="0" cellspacing="0" style="width:356px;">
			<tbody>
				<tr height="22">
					<td colspan="3" height="22" style="height:22px;width:356px;">Plasmid mix</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;"></td>
					<td>per well</td>
					<td>per 96-well plate</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;">MEM</td>
					<td>--</td>
					<td>500 &#181;l</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;">RTP1S-pCI</td>
					<td>5 ng</td>
					<td>480 ng</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;">M3-R-pCI</td>
					<td>2.5 ng</td>
					<td>240 ng</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;">pCRE-luc</td>
					<td>10 ng</td>
					<td>960 ng</td>
				</tr>
				<tr height="22">
					<td height="22" style="height:22px;">pSV40-RL</td>
					<td>5 ng</td>
					<td>480 ng</td>
				</tr>
			</tbody>
		</table> 
Table 1. Plasmid mix components. Per well and per 96-well plate volumes of RTP1S-pCI, M3-R-pCI, pCRE-luc, and pSV40-RL, and MEM.
		</li>
		<li>For each 96-well plate, dilute 18 &#181;l lipid transfection reagent in 450 &#181;l MEM medium.</li>
		<li>Pipette Plasmid mix (from Step 3.3.1), rhodopsin-tagged olfactory receptor in pCI plasmid (Rho-OR-pCI), and lipid transfection mix (from Step 3.3.2) to make the Complex detailed in Table 2. Mix the solution by trituration and incubate at room temperature for 15 min. Stop the reaction by adding M10 according to Table 2. NOTE: this reaction is time-sensitive and should not be allowed to continue for more than 30 min. The well + 10% calculation is important to ensure sufficient volume for subsequent steps.
		<table border="0" cellpadding="0" cellspacing="0">
			<tbody>
				<tr height="21">
					<td colspan="3" height="21" style="height:21px;width:472px;">Complex</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;"></td>
					<td>per well</td>
					<td>per well + 10%</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;">Plasmid mix</td>
					<td>4.2 &#181;l</td>
					<td>4.58 &#181;l</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;">Rho-OR-pCI</td>
					<td>0.05 ng</td>
					<td>0.06 ng</td>
				</tr>
				<tr height="21">
					<td height="21" style="height:21px;">Lipid transfection mix</td>
					<td>4.2 &#181;l</td>
					<td>4.58 &#181;l</td>
				</tr>
				<tr height="22">
					<td height="22" style="height:22px;">M10</td>
					<td>41.7 &#181;l</td>
					<td>45.83 &#181;l</td>
				</tr>
			</tbody>
		</table> 
Table 2. Complex components. Per well and per well + 10% volumes of plasmid mix (Table 1), olfactory receptor plasmid (Rho-OR-pCI), and lipid transfection mix. M10 is added to quench the reaction following a 15 min&#160;incubation at room temperature.
		</li>
	</ol>
	</li>
</ol>
<ol start="4">
	<li>Tap out the media on the cell plates.</li>
	<li>Pipette 50 &#181;l of complex to each well and incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
4. Odor Stimulation
<ol>
	<li>Observe the transfected cells to ensure a proper confluency of 50-80% per well and return to incubator. NOTE: If cells are less than 50% confluent, firefly luciferase and renilla luciferase readings may be too low for measurement of receptor activation. Consider discarding the plate.</li>
	<li>Prepare 1 M stock solutions of each odor in DMSO.</li>
	<li>Prepare odor stimulation solutions in CD293 medium.
	<ol>
		<li>For screening experiments, dilute stock solution of odor to 100 &#181;M. Also prepare a no-odor control (CD293 only) in order to control for OR background activation. NOTE: For screening experiments, each OR/odor pair is tested only once per experiment. Because some odor diffusion across wells is possible, it is recommended to stimulate with one odorant for each plate.</li>
		<li>For dose response experiments, prepare seven 10-fold serial dilutions of odor stock solution in triplicate starting at 1 mM for each receptor. Also prepare the same odor dilutions in triplicate for empty vector-transfected cells in order to control for odor background activation. NOTE: For dose response experiments, each odor concentration treatment should be conducted in triplicate.</li>
	</ol>
	</li>
	<li>Tap out the media on the cell plates.</li>
	<li>Pipette 25 &#181;l of odor stimulation solution to each well and incubate for 4 hr.</li>
</ol>
5. Measuring OR Activity via Luciferase Assay
<ol>
	<li>Resuspend firefly luciferase substrate per the manufacturer&#8217;s instructions and store at -80 &#176;C.</li>
	<li>Thaw 1 ml of firefly luciferase substrate per 96-well plate.</li>
	<li>Prepare fresh firefly luciferase reaction quencher and Renilla luciferase substrate reagent (5 &#181;l luciferase quencher/Renilla luciferase substrate per 1 ml of buffer). NOTE: Approximately 1 ml of reagent is needed per 96-well plate.</li>
	<li>Prepare the luminescent microplate reader. Open the microplate reader software. Within the system icon:
	<ol>
		<li>Under the &#34;Pre-heating&#34; Tab, check the box for &#34;ON&#34; and set the temperature of the machine to 25 &#176;C.</li>
		<li>Under the &#34;Dispenser&#34; tab, prime each dispenser with 1,000 &#181;l of 70% ethanol followed by 1,000 &#181;l of distilled water. NOTE: Use separate aliquots of alcohol and water for each dispenser. Ethanol is used to disinfect the dispensers, and water removes the residual ethanol.</li>
		<li>Prime each dispenser with 1,500 &#181;l of air (remove dispensers from liquid). NOTE: priming with air ensures that luciferase substrates are not diluted with residual water.</li>
		<li>Prime dispenser 1 with 1,080 &#181;l of firefly luciferase substrate (from Step 5.2). Prime dispenser 2 with Renilla luciferase substrate (from Step 5.3). NOTE: Be careful not to cross-contaminate the luciferase substrates. Priming with luciferase substrates fills the dead space in the reagent dispensers.</li>
	</ol>
	</li>
	<li>Set up the following protocol to read both firefly and Renilla luciferase luminescence. Within the software associated with the microplate reader, under the &#34;File&#34; menu, click on &#34;New Task&#34;. Highlight &#34;Protocols&#34; and click on &#34;Create New&#34;. In the next window, the circle next to &#34;Standard Protocol&#34; should be selected. Click &#34;OK.&#34; Double click on &#34;Procedure&#34; on the left hand side of the screen.
	<ol>
		<li>Dispense 10 &#181;l of firefly luciferase substrate to all wells using dispenser 1. Under the &#34;Actions&#34; menu, click &#34;Dispense&#34;. In the &#34;Dispense Step&#34; window, set: &#34;Dispenser&#34; to 1, &#34;Priming&#34; to none, &#34;Dispense Volume&#34; to 10 &#181;l and &#34;Rate&#34; to 225 &#181;l/sec. Click &#34;OK&#34;.</li>
		<li>Shake the plate for 30 sec. Under the &#34;Actions&#34; menu, click &#34;Shake&#34;. In the &#34;Shake Step&#34; window, set &#34;Intensity&#34; to Medium and &#34;Duration&#34; to 0:30 MM:SS. Click &#34;OK&#34;.</li>
		<li>Read the luminescence of all wells for 0.5 sec per well. Under the &#34;Actions&#34; menu, click &#34;Read&#34;. In the &#34;Read Step&#34; window, set: &#34;Detection Method&#34; to Luminescence, &#34;Read Type&#34; to Endpoint, &#34;Integration Time&#34; to 0:00:50 MM:SS:ss, &#34;Filter Sets&#34; to 1, &#34;Emission&#34; to Hole, &#34;Optics Position&#34; to Top, &#34;Gain&#34; to 135, and &#34;Read Height&#34; to 1.00 mm. Click &#34;OK&#34;.</li>
		<li>Dispense 10 &#181;l of Renilla luciferase substrate to all wells using dispenser 2. Set the conditions as in Step 5.6.1, except set &#34;Dispenser&#34; to 2.</li>
		<li>Shake plate for 30 sec. Set the conditions as in Step 5.6.2.</li>
		<li>Read luminescence of all wells for 0.5 sec per well. Set the conditions as in Step 5.6.3.</li>
	</ol>
	</li>
	<li>Remove the lid from the 96-well plate and place the plate in the microplate reader. Start the program set up in Step 5.5 to read plate luminescence.</li>
	<li>Clean the reagent dispenser pumps. From the system icon under the &#34;Dispenser&#34; tab:
	<ol>
		<li>Purge 1,000 &#181;l of firefly luciferase substrate from the firefly luciferase dispenser into a recovery tube. NOTE: Firefly luciferase can be stored at -80 &#176;C and reused.</li>
		<li>Prime each dispenser with 1,000 &#181;l of distilled water, followed by 1,000 &#181;l of 70% ethanol, and finally 1,500 &#181;l of air (remove dispensers from liquid). NOTE: Water removes luciferase substrates from the reagent pumps, ethanol disinfects, and air dries any residual ethanol.</li>
	</ol>
	</li>
</ol>
6. Data Analysis
<ol>
	<li>Data Export
	<ol>
		<li>In the microplate reader software, double click on &#34;Report/Export Builders&#34; on the left side of the screen.</li>
		<li>Click on the button &#34;New Export to Excel&#34; and click &#34;OK&#34;.</li>
		<li>Highlight Export1 and click &#34;Edit&#34;.
		<ol>
			<li>Under &#34;Content&#34; check &#34;System Description&#34;, &#34;Procedure&#34;, &#34;Plate Description&#34;, and &#34;Plate Layout Matrix&#34;. Include &#34;Raw Data&#34; and &#34;Calculated Data&#34;.</li>
			<li>Under &#34;Workflow&#34;, check &#34;Autoexecute on completion of the procedure&#34;. Under Export Mode, check &#34;All plates in the same workbook&#34; and &#34;As a new worksheet&#34;.</li>
			<li>Under &#34;File&#34; choose file name format and file location, and click &#34;OK&#34;.</li>
			<li>Close the &#34;Report/Export Builders&#34; window.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>To obtain normalized luciferase values, divide the firefly luciferase luminescence reading for each well (Step 5.6.3) by the Renilla luminescence reading for each well (Step 5.6.6).</li>
</ol>

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K., Mori, K., Harel, D., Sobel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metric+for+odorant+comparison.">A metric for odorant comparison.</a> Nature methods. 5 (5), 425-429 (2008).</li><li>Veithen, A., Wilkin, F., Philippeau, M., Van Osselaer, C., Chatelain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+Receptors:+From+basic+science+to+applications+in+flavors+and+fragrances.">Olfactory Receptors: From basic science to applications in flavors and fragrances.</a> Perfumer and Flavorist. 35 (1), 38-40 (2010).</li><li>Bozza, T., Feinstein, P., Zheng, C., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptor+expression+defines+functional+units+in+the+mouse+olfactory+system.">Odorant receptor expression defines functional units in the mouse olfactory system.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 22 (8), 3033-3043 (2002).</li><li>Oka, Y., Katada, S., Omura, M., Suwa, M., Yoshihara, Y., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptor+map+in+the+mouse+olfactory+bulb:+in+vivo+sensitivity+and+specificity+of+receptor-defined+glomeruli.">Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity and specificity of receptor-defined glomeruli.</a> Neuron. 52 (5), 857-869 (2006).</li><li>Zhao, H., Ivic, L., Otaki, J. M., Hashimoto, M., Mikoshiba, K., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+a+mammalian+odorant+receptor.">Functional expression of a mammalian odorant receptor.</a> Science. 279 (5348), 237-242 (1998).</li><li>Touhara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+identification+and+reconstitution+of+an+odorant+receptor+in+single+olfactory+neurons.">Functional identification and reconstitution of an odorant receptor in single olfactory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 4040-4045 (1999).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+of+accessory+factors+in+functional+expression+of+mammalian+odorant+receptors.">Synergism of accessory factors in functional expression of mammalian odorant receptors.</a> The Journal of biological chemistry. 282 (20), 15284-15293 (2007).</li><li>McRae, J. F., Mainland, J. D., Jaeger, S. R., Adipietro, K. A., Matsunami, H., Newcomb, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+the+odorant+receptor+OR2J3+is+associated+with+the+ability+to+detect+the+&amp;#34;grassy&amp;#34;+smelling+odor,+cis-3-hexen-1-ol.">Genetic variation in the odorant receptor OR2J3 is associated with the ability to detect the &amp;#34;grassy&amp;#34; smelling odor, cis-3-hexen-1-ol.</a> Chemical senses. 37 (7), 585-593 (2012).</li><li>Adipietro, K. A., Mainland, J. D., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+evolution+of+mammalian+odorant+receptors.">Functional evolution of mammalian odorant receptors.</a> PLoS genetics. 8 (7), (2012).</li><li>Zhuang, H., Chien, M. -. S., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+functional+evolution+of+an+odorant+receptor+for+sex-steroid-derived+odors+in+primates.">Dynamic functional evolution of an odorant receptor for sex-steroid-derived odors in primates.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (50), 21247-21251 (2009).</li><li>Oka, Y., Nakamura, A., Watanabe, H., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+odorant+derivative+as+an+antagonist+for+an+olfactory+receptor.">An odorant derivative as an antagonist for an olfactory receptor.</a> Chemical senses. 29 (9), 815-822 (2004).</li><li>Oka, Y., Omura, M., Kataoka, H., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+receptor+antagonism+between+odorants.">Olfactory receptor antagonism between odorants.</a> The EMBO journal. 23 (1), 120-126 (2004).</li><li>Fawcett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+ROC+analysis.">An introduction to ROC analysis.</a> Pattern Recognition Letters. 27 (8), 861-874 (2006).</li><li>Baghaei, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+Receptors.">Olfactory Receptors.</a> Olfactory Recept. Methods Protoc. 1003, 229-238 (2013).</li><li>Dey, S., Zhan, S., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+surface+expression+of+chemosensory+receptors+in+heterologous+cells.">Assaying surface expression of chemosensory receptors in heterologous cells.</a> Journal of visualized experiments JoVE. (48), (2011).</li></ol>

The authors have nothing to disclose.&#160;

Odorant identity is encoded by olfactory receptor activation patterns, but receptor activation patterns, including which receptors are activated and to what degree, are known for fewer than 6% of human olfactory receptors1-11. Efforts to characterize olfactory receptors have been limited by their labor-intensive methods or applicability to only a subset of the olfactory receptor family17,23,24,33,34. The Hana3A heterologous expression system supports the robust expression of the majority of tested o...

The mammalian olfactory system has the ability to respond to a vast number of odorous stimuli, allowing for the detection and discrimination of thousands of odorants. Olfactory receptors (ORs) are the molecular sensors expressed by the olfactory sensory neurons in the olfactory epithelium12. Mammalian odor recognition occurs through differential activation of ORs by odorants, and the OR gene family is extensive, with roughly 1,000 murine and 400 human receptors12-16. Previous functional analyses of ORs in olfactory neurons and in heterologous cells have shown that different odorants are recognized by unique, but overlapping ensembles of ORs10,17-20. Matching ligands to ORs is critical for understanding the olfactory code and essential for building viable models of olfaction. Due to difficulties expressing ORs in heterologous systems as well as the large number of both odorants and ORs, this data has been largely absent from the field; indeed, fewer than 6% of human ORs have a published ligand1-11. This protocol describes the use of a luciferase assay to characterize odorant/OR interactions. This assay enables the high-throughput characterization of ORs, a task that is essential to understanding odorant/OR interactions as well as developing a model of odor coding.
High-throughput studies of ORs face three major challenges. First, ORs expressed in heterologous cells were retained in the ER and subsequently degraded in the proteasome21,22, preventing the ORs from interacting with odorants in the assay system23-25. This problem was addressed by the discovery of accessory proteins that facilitate stable cell-surface expression of a broad range of ORs19,26,27. Receptor-transporter-proteins 1 and 2 (RTP1 and 2) promote OR cell-surface expression and activation in response to odorant stimulation19. Based on this work, HEK293T cells were modified to stably express RTP1 long (RTP1L) and RTP2, receptor expression-enhancing protein 1, and G&#945;olf, resulting in the Hana3A cell line19,27. In addition, the type 3 muscarinic acetylcholine receptor (M3-R) interacts with ORs at the cell surface and enhances activation in response to odorants26. Co-transfection of an OR with RTP1S and M3-R into Hana3A cells results in the robust, consistent, and functional expression of a broad range of ORs at the cell surface27. Second, mammalian OR repertoires are quite large. In humans, for example, the OR repertoire is an order of magnitude more numerous than the gustatory receptor repertoire, and 2&#160;orders of magnitude more numerous than the visual receptor repertoire. Although cloning a single OR is a relatively straightforward protocol, significant up-front effort is required to generate a comprehensive library. Third, although we know that in vision, wavelength translates into color and in audition frequency translates into pitch, the organization of odors is poorly understood, making it difficult for researchers to interpolate from a representative sample of odorants. Although some progress has been made on this front10,28, the map of the olfactory landscape remains incomplete. Screening tens of thousands of molecules against hundreds of ORs is a daunting task; high-throughput screens in this domain require carefully defined campaigns. The major remaining challenges are those of logistics and cost rather than problems inherent to the technique. Although heterologous screening has not been widely used to identify ligands by academic groups, a private company has used the same technique to identify ligands for 100 human ORs 29. Unfortunately, these data remain proprietary.
The high-throughput luciferase assay outlined here has several advantages over alternative methods used to assess OR activation. Although the responses of native olfactory sensory neurons have been measured using electrophysiology and calcium imaging, these techniques have difficulty teasing apart which OR leads to a neuron&#39;s response due to the overlap in response properties for olfactory neurons. Although knocking-in a GFP-labeled receptor type30,31, delivering specific receptors via adenovirus to murine olfactory neurons32,33, or performing RT-PCR after recordings17,24,33 can link recordings to single receptor types, these methods are low-throughput and not suitable for large-scale screens. Heterologous screening systems are more scalable, and two major forms are found in the literature: cAMP pathway reporters and inositol triphosphate (IP3) pathway reporters. Upon odor stimulation, ORs activate a G&#945;olf transduction signaling cascade that results in the production of cyclic AMP (cAMP)12. By co-transfecting a firefly luciferase reporter gene under the control of a cAMP response element (CRE), luciferase production can be measured as a function of odor response, allowing for the quantification of OR activation. OR activation can also be linked to the IP3 pathway by co-expressing G-proteins such as G&#945;15/16 or a G&#945;15-olf chimera24,25,34. We have chosen the assay presented here based on three factors: (1) the co-expression of RTP1 with Rho-tagged olfactory receptors improves the expression of olfactory receptors at the cell surface19,27; (2) use of a cAMP-responsive reporter gene allows for the measurement of OR activation through the canonical second messenger pathway; and (3) the assay is well-suited to high-throughput screens.
This high-throughput luciferase assay is applicable to a variety of studies valuable to the field of olfaction. First, a large number of ORs can be screened against a single odorant in order to determine the receptor activation pattern for a specific odorant. This type of study identified OR7D4 as the OR responsible for responding to the steroid odorant androstenone8. Conversely, one OR can be screened against a panel of odorants in order to determine the receptor response profile10. When candidate olfactory odorant/OR pairs are identified via these screens, interaction can be confirmed by conducting a dose response experiment examining the response of the OR to increasing concentrations of odorant. Dose response curves can also assess how genetic variation in an OR affects in vitro odorant response8,9,11,35, and these studies can be extended to interspecific OR variation, allowing for the examination of receptor evolution across species and causal mutations in evolution36,37, Finally, this assay can be used to screen for odor antagonists that are able to antagonize OR response to a particular odorant for a known odorant/receptor pair38,39. In summary, this high-throughput luciferase assay is applicable to a range of studies that will help characterize OR activation patterns and provide a better understanding of odor coding in the olfactory system.

<table border="0" cellpadding="0" cellspacing="0" style="width:685px;" width="685">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">Hana3A cells</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">RTP1S-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">M3-R-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pCRE-luc</td>
			<td style="width:135px;">Agilent</td>
			<td style="width:119px;">219076</td>
			<td><a href="http://www.chem.agilent.com/store/ProductDetail.aspx?productID=219076%28Agilent%29">LUC</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pSV40-RL</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2231</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-vectors-and-cell-lines/prl-renilla-luciferase-control-reporter-vectors/">RL</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Media, Eagle</td>
			<td>Sigma Aldrich</td>
			<td>M4655</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m4655?lang=en&amp;region=US">MEM</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">FBS</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/16000044?ICID=search-16000044">FBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (without Ca2+ and Mg2+)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td><a href="http://cellgro.com/products/balanced-salt-solutions/phosphate-buffered-saline/phosphate-buffered-saline-1x-2.html">PBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% Trypsin EDTA)</td>
			<td>Life Technologies</td>
			<td>25300</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product">Trypsin</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">CD293</td>
			<td>Life Technologies</td>
			<td>11913-019</td>
			<td><a href="http://products.invitrogen.com/ivgn/product/11913019?ICID=search-11913019">CD293</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">96 well PDL white/clear plate</td>
			<td style="width:135px;">BD BioCoat</td>
			<td style="width:119px;">356693</td>
			<td><a href="http://www.thomassci.com/Supplies/Tissue-Culture-Cellware/_/BD-Biocoat-Poly-D-Lysine-Culture-Dishes?q=356693">plates</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Lipid transfection reagent: Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/11668019?ICID=search-11668019">Lipofectamine</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:265px;">Firefly luciferase substrate, firefly luciferase quencher/Renilla luciferase substrate: Dual-Glo Assay</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2980</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_glo-luciferase-assay-system/?__utma=1.1932750276.1376486173.1376486173.1376486173.1&amp;__utmb=1.1.10.1376486173&amp;__utmc=1&amp;__utmx=-&amp;__utmz=1.1376486173.1.1.utmcsr=google|utmc">dual glo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy S2&#160;</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">SLAD</td>
			<td><a href="http://www.biotek.com/products/microplate_detection/synergy2_multimode_microplate_reader.html">BioTek S2</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Microplate reader software: Gen5 Data Analysis Software</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">Gen5</td>
			<td><a href="http://www.biotek.com/products/microplate_software/gen5_data_analysis_software.html">Gen5</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">BIOSTACK</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">BIOSTACK2WR</td>
			<td><a href="http://www.biotek.com/products/microplate_software/biostack_microplate_stacker.html">BioStack</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Multiflo</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">MFP</td>
			<td><a href="http://www.biotek.com/products/liquid_handling/multiflo_microplate_dispenser.html">MultiFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4414</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips ViaFlo96</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips 384 XYZ</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6403</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">384ViaFlo</td>
			<td style="width:135px;">Integra</td>
			<td>6030</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/96_384_channel_pipette.html">384ViaFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td>Macherey Nagel</td>
			<td>740797.1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ML</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d2650?lang=en&amp;region=US">DMSO</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forskolin</td>
			<td>Enzo Life Sciences</td>
			<td>BML-CN100-0010</td>
			<td><a href="http://www.thomassci.com/007635F0-AA41-4B24-B46A-05723D685997/_/FORSKOLIN-10MG?q=c002e24">FOR</a></td>
		</tr>
	</tbody>
</table>

high-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

A primary screen tested 328 ORs against 26 odors at a concentration of 100 &#181;M. This odor concentration has been demonstrated to effectively activate a large proportion of ORs with known ligands10. First, normalized luciferase activity was calculated by dividing the firefly luciferase reading by the Renilla luciferase reading. Next, baselined values were calculated by subtracting the normalized luciferase readings for the no odor control from the normalized luciferase readings for each well (<stro...

Watch this Scientific Journal Video about High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity at JoVE.com

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

high-throughput-analysis-mammalian-olfactory-receptors-measurement

Research

JoVE Journal

Neuroscience

12.4K Görüntüleme.  Monell Chemical Senses Center.  Koku reseptör aktivasyonu desenler koku kimliğini kodlamak, ancak memeli koku reseptörleri için Odorant ligandlar belirlenmesi yayınlanan verilerin eksikliği koku kodlama kapsamlı bir modelinin gelişmesini engellemektedir. Bu protokol, koku reseptör ligandları ve reseptör aktivasyonunun miktar yüksek throughput belirlenmesini kolaylaştırmak için bir yöntem tarif etmektedir. 

Video: High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.